Gail L. Kurriger

A mouse model of osteochondromagenesis from clonal inactivation of Ext1 in chondrocytes

We report a mouse model of multiple osteochondromas (MO), an autosomal dominant disease in humans, also known as multiple hereditary exostoses (MHE or HME) and characterized by the formation of cartilage-capped osseous growths projecting from the metaphyses of endochondral bones. The pathogenesis of these osteochondromas has remained unclear. Mice heterozygous for Ext1 or Ext2, modeling the human genotypes that cause MO, occasionally develop solitary osteochondroma-like structures on ribs [Lin et al. (2000) Dev Biol 224(2):299–311; Stickens et al. (2005) Development 132(22):5055–5068]. Rather than model the germ-line genotype, we modeled the chimeric tissue genotype of somatic loss of heterozygosity (LOH), by conditionally inactivating Ext1 via head-to-head loxP sites and temporally controlled Cre-recombinase in chondrocytes. These mice faithfully recapitulate the human phenotype of multiple metaphyseal osteochondromas. We also confirm homozygous disruption of Ext1 in osteochondroma chondrocytes and their origin in proliferating physeal chondrocytes. These results explain prior modeling failures with the necessity for somatic LOH in a developmentally regulated cell type.

CD44 expression in the developing and growing rat intervertebral disc

CD44 has been identified at the time of extracellular matrix formation and expansion in several sites of the developing embryo (Wheatley et al. [ 1993 ] Development 119:295–306). The nucleus pulposus, consisting of a hydrated extracellular matrix tissue at birth, not previously closely analyzed, was examined for expression of CD44 in the developing and aging rat intervertebral disc. CD44 was identified solely on notochordal cells from the first onset of intervertebral disc formation (day 15 embryo) through the loss of notochordal cells from the nucleus pulposus (12–24 months of age). No CD44 expression was found in the notochordal cells prior to disc formation or in any cells other than the notochordal cells in the annulus fibrosus or nucleus pulposus of the intervertebral disc. Using reverse transcriptase–polymerase chain reaction methodology, the single 365 amino acid CD44 standard, CD44s, open reading frame was amplified from notochordal cells isolated from the nucleus pulposus. Western blot analysis of a cultured nucleus pulposus notochordal cells total protein extract identified a single CD44 species devoid of chondroitin sulfate with a mass of ∼85 kDa, characteristic of CD44s. Cell surface detection for CD44 was co‐localized with hyaluronan and proteoglycans at first appearance of disc formation in the nucleus pulposus.

Rat spinal motion segment in organ culture: a cell viability study.

Study Design. This study investigated tissue integrity and viability of cells in an organ culture system of intervertebral disc (IVD) with adjoining vertebral bodies. Objective. The goal of this study was to design a methodology to maintain an IVD motion segment in organ culture, thereby preserving viability and tissue architecture. Summary of Background Data. Study of IVD mechanobiology in vitro necessitates availability of vertebral bodies for controlled application of complex loads. Methods. IVD motion segments were dissected from rat lumbar segments and maintained in organ culture and cell viability was evaluated histochemically using NitroBlue Tetrazolium. Tissue integrity and morphology were evaluated using conventional histologic techniques. Results. The in vitro organ culture of motion segments maintained the viability and tissue integrity for 14 days. More than 95% viability in all three regions of interest (anulus fibrosus, nucleus pulposus, end plates) was maintained for 14 days in culture. Conclusion. Our initial results suggest that long-term motion segment culture is practical, and the inclusion of vertebral bodies will facilitate anchoring during biomechanical stimulation. Thus, we expect the culture system to provide us with an excellent model for studying the pathomechanics of IVD degeneration and the effects of mechanical stimulation on the biology of IVD cells.

Comparative digital cartilage histology for human and common osteoarthritis models

PURPOSE This study addresses the species-specific and site-specific details of weight-bearing articular cartilage zone depths and chondrocyte distributions among humans and common osteoarthritis (OA) animal models using contemporary digital imaging tools. Histological analysis is the gold-standard research tool for evaluating cartilage health, OA severity, and treatment efficacy. Historically, evaluations were made by expert analysts. However, state-of-the-art tools have been developed that allow for digitization of entire histological sections for computer-aided analysis. Large volumes of common digital cartilage metrics directly complement elucidation of trends in OA inducement and concomitant potential treatments. MATERIALS AND METHODS Sixteen fresh human knees, 26 adult New Zealand rabbit stifles, and 104 bovine lateral plateaus were measured for four cartilage zones and the cell densities within each zone. Each knee was divided into four weight-bearing sites: the medial and lateral plateaus and femoral condyles. RESULTS One-way analysis of variance followed by pairwise multiple comparisons (Holm-Sidak method at a significance of 0.05) clearly confirmed the variability between cartilage depths at each site, between sites in the same species, and between weight-bearing articular cartilage definitions in different species. CONCLUSION The present study clearly demonstrates multisite, multispecies differences in normal weight-bearing articular cartilage, which can be objectively quantified by a common digital histology imaging technique. The clear site-specific differences in normal cartilage must be taken into consideration when characterizing the pathoetiology of OA models. Together, these provide a path to consistently analyze the volume and variety of histologic slides necessarily generated by studies of OA progression and potential treatments in different species.

Biocompatibility and preclinical feasibility tests of a temperature-sensitive hydrogel for the purpose of surgical wound pain control and cartilage repair

We recently introduced a novel pluronic F127 and hyaluronic acid-based hydrogel (HG) designed to deliver a broad range of therapeutics. The reverse-thermal responsive HG exhibits physical properties that seem to be ideal for the local delivery of drug- and cell-based therapies to specific anatomic sites through percutaneous injection. However, questions related to the HGs safety and efficacy must first be addressed. To address these issues, we performed standard in vitro cytotoxicity and drug release tests and in vivo biocompatibility tests in a rat model. In addition, we determined whether the HG was an effective stem cell carrier in a rat cartilage defect model. We found that the HG showed viability and biocompatibility levels similar to those reported for F127 or hyaluronic acid alone. In vitro drug release studies with bupivacaine, a drug used clinically for local pain relief, revealed that after an initial burst bupivacaine was released continuously for 10 days. Stem cells loaded in the HG were retained in situ and stimulated cartilage regeneration in experimental defects. Taken as a whole, these findings support further efforts to develop the HG as a versatile system for the delivery of a wide range of therapeutic agents in humans.

Telomerase Reverse Transcriptase Subunit Expression Is Associated with Chondrosarcoma Malignancy

Expression of the telomerase reverse transcriptase subunit telomerase reverse transcriptase gene is associated with most human malignancies. Because telomerase reverse transcriptase is rarely expressed in normal tissue, its presence in pathologic specimens is considered a marker of transformed cells. Moreover, high levels of expression have been correlated with poor prognosis in many cancers. Although telomerase activity has been found in chondrosarcomas, its prognostic significance in these malignant cartilage tumors is unknown. Malignancy in cartilage-derived tumors is assessed routinely by histomorphologic grading, but even well differentiated, low-grade lesions can metastasize. This unpredictable behavior greatly complicates the clinical treatment of cartilage tumors, making better prognostic indicators desirable. To address this issue we used immunohistochemistry to compare telomerase reverse transcriptase expression in a collection of 61 tumors consisting of malignant chondrosarcomas of varying grade and benign enchondromas. Associated case histories were reviewed to test the hypothesis that telomerase reverse transcriptase expression levels correlated with subsequent tumor recurrence. We found that the relative abundance of telomerase reverse transcriptase-expressing cells correlated significantly with grade and recurrence. These findings indicate that telomerase reverse transcriptase immunostaining may be a useful adjunct to the conventional three-level grading system.

Organ culture stability of the intervertebral disc: Rat versus rabbit

There is a need to develop mechanically active culture systems to better understand the role of mechanical stresses in intervertebral disc (IVD) degeneration. Motion segment cultures that preserve the native IVD structure and adjacent vertebral bodies are preferred as model systems, but rapid ex vivo tissue degeneration limits their usefulness. The stability of rat and rabbit IVDs is of particular interest, as their small size makes them otherwise suitable for motion segment culture. The goal of this study was to determine if there are substantial differences in the susceptibility of rat and rabbit IVDs to culture‐induced degeneration. Lumbar IVD motion segments were harvested from young adult male Sprague–Dawley rats and New Zealand White rabbits and cultured under standard conditions for 14 days. Biochemical assays and safranin‐O histology showed that while glycosaminoglycan (GAG) loss was minimal in rabbit IVDs, it was progressive and severe in rat IVDs. In the rat IVD, GAG loss was concomitant with the loss of notochordal cells and the migration of endplate (EP) cells into the nucleus pulposus (NP). None of these changes were evident in the rabbit IVDs. Compared to rabbit IVDs, rat IVDs also showed increased matrix metalloproteinase‐3 (MMP‐3) and sharply decreased collagen type I and II collagen expression. Together these data indicated that the rabbit IVD was dramatically more stable than the rat IVD, which showed culture‐related degenerative changes. Based on these findings we conclude that the rabbit motion segments are a superior model for mechanobiologic studies.

Biomechanical disc culture system: feasibility study using rat intervertebral discs.

A small-scale biomechanical disc culture system was designed to stimulate intervertebral disc (IVD) ‘motion segment’ in culture environment with load-controlled compression and combined load (compression + shear). After 7 days of diurnal mechanical loading, cell viability of discs stimulated with static compression load (0.25 MPa) and static combined load (compression (0.25 MPa) + shear (1.5 N)) were similar (> 90 per cent) to unloaded controls. Mechanically stimulated discs showed decrease in static/dynamic moduli, early stress relaxation, and loss of disc height after 7 days of diurnal loading. Histological data of discs indicated load-induced transformations that were not apparent in controls. The feasibility of studying the mechanobiology of intact IVD as a motion segment was demonstrated. Media conditioning (improve tissue stability in long-term culture) and application of biochemical gene expression assays (differential tissue response to types of mechanical stimulation) are proposed as future improvements. The study suggests that the limitations in studying mechanobiology of IVD pathology in vitro can be overcome and it is possible to understand the physiologically relevant mechanism of IVD pathology.

Effects of knockout of the receptor for advanced glycation end-products on bone mineral density and synovitis in mice with intra-articular fractures: RAGE KNOCKOUT EFFECTS IN MICE WITH JOINT FRACTURES

Our group employed the mouse closed intra‐articular fracture (IAF) model to test the hypothesis that the innate immune system plays a role in initiating synovitis and post‐traumatic osteoarthritis (PTOA) in fractured joints. A transgenic strategy featuring knockout of the receptor for advanced glycation end‐products (RAGE −/−) was pursued. The 42 and 84 mJ impacts used to create fractures were in the range previously reported to cause PTOA at 60 days post‐fracture. MicroCT (μCT) was used to assess fracture patterns and epiphyseal and metaphyseal bone loss at 30 and 60 days post‐fracture. Cartilage degeneration, synovitis, and matrix metalloproteinase (MMP‐3, ‐13) expression were evaluated by histologic analyses. In wild‐type mice, μCT imaging showed that 84 mJ impacts led to significant bone loss at 30 days (p < 0.05), but recovered to normal at 60 days. Bone losses did not occur in RAGE−/− mice. Synovitis was significantly elevated in 84 mJ impact wild‐type mice at both endpoints (30 day, p = 0.001; 60 day, p = 0.05), whereas in RAGE−/− mice synovitis was elevated only at 30 days (p = 0.02). Mankin scores were slightly elevated in both mouse strains at 30 days, but not at 60 days. Immunohistochemistry revealed significant fracture‐related increases in MMP‐3 and −13 expression at 30 days (p < 0.05), with no significant difference between genotypes. These findings indicated that while RAGE −/− accelerated recovery from fracture and diminished synovitis, arthritic changes were temporary and too modest to detect an effect on the pathogenesis of PTOA.